PROJECT SUMMARY: Osteoporosis is the most common metabolic bone disease mainly characterized by low bone mineral density (BMD) and deteriorated bone quality/strength. Peripheral blood monocytes (PBMs) may not only act as precursors of osteoclasts but also produce cytokines important for osteoclast differentiation and function, and thus represent major systemic cells for bone metabolism. DNA methylation as an important epigenetic regulator of gene expression may have significant and potentially sex-specific effects in the etiology of human complex diseases. However, the significance of global DNA methylation profiles underlying osteoporosis risk is largely unknown, particularly in males who suffer significantly higher mortality rate upon osteoporotic fractures than females. Our Hypothesis is that altered DNA methylation profiles in PBMs and the associated changes in gene expression and osteoclastogenesis contribute to variations in peak BMD and bone quality/strength in males. Our Goal/Expectation is to i) identify significant differentially methylated regions (DMRs) in PBMs associated with osteoporosis risk in Caucasian males; ii) assess the potential sex- and ethnic-generality/specificity of the significant DMRs; and iii) ascertain the DNA methylation mediated epigenetic mechanisms of osteoporosis, that is, how the DMRs regulate the expression of the target genes and subsequent osteoclastogenesis. We will accomplish the following Specific Aims: 1) Identification and validation of DMRs significantly associated with peak BMD and bone quality/strength (QCT and FEA) in Caucasian males. We will perform PBM methylome profiling analyses with double restriction-enzyme reduced representation bisulfite sequencing (dRRBS) assays in 200 discordant Caucasian males (?Discovery cohort?) at peak bone mass ages of 20-30 years old, including half with high peak BMDs and the other half with low peak BMDs, and validate the most significant DMRs in both of the ?Discovery cohort? and an independent ?Replication cohort? of 200 Caucasian males discordant for peak BMDs. 2) Evaluation of the sex-, ethnic-, and maturation stage- generality/specificity of the significant DMRs/genes. The validated DMRs/target genes will be tested in three independent BMD-discordant samples, including a) 200 African American males, b) 160 Caucasian females, and c) 160 Chinese males, and d) in 1670 US children from the Bone Mineral Density in Childhood Study. 3) In-depth functional investigation of the roles of DMRs in regulating gene expression and osteoclastogenesis. We will identify the DMR-regulated target genes by correlating the DNA methylation and the mRNA expression levels of candidate target genes in the same sets of PBMs from the total 400 Caucasian males, and conduct in vitro cell-based assays to determine the contribution of DNA methylation at these DMRs in regulating target gene expression and subsequently influencing osteoclastogenesis. The results will reveal novel and fundamental insights into the general and sex-/ethnic-/development specific epigenetic mechanisms underlying osteoporosis. The knowledge gained may ultimately lead to novel approaches to better prevention and treatment of osteoporosis.